Factor VIII proteins having ancestral sequences, expression vectors, and uses related thereto

Abstract

This disclosure relates to recombinant or chimeric FVIII proteins, variants, and vectors encoding the proteins containing one or more ancestral mutations. In certain embodiments, one or more protein domains comprise amino acid sequences that are derived from ancestrally reconstructed amino acid sequences. In certain embodiments, the disclosure relates to pharmaceutical compositions comprising the proteins or vectors and related methods of inducing blood clotting.

The invention claimed is: 
     
       1. A recombinant or chimeric FVIII protein, comprising, in an N- to C-terminal direction, an A1 domain, an A2 domain, a linker, an ap domain, an A3 domain, a C1 domain, and a C2 domain, wherein the protein has a B-domain deletion with the A2 and ap domains joined by the linker, and wherein the protein has FVIII activity, and:
 the A1 domain has an amino acid sequence at least 96% identical to SEQ ID NO: 22; 
 the A2 domain has an amino acid sequence at least 96% identical to SEQ ID NO: 23; 
 the ap domain has an amino acid sequence at least 96% identical to SEQ ID NO: 25; 
 the A3 domain has an amino acid sequence at least 96% identical to SEQ ID NO: 26; 
 the C1 domain has an amino acid sequence at least 96% identical to SEQ ID NO: 27; and 
 the C2 domain has an amino acid sequence at least 96% identical to SEQ ID NO: 28. 
 
     
     
       2. The protein of  claim 1 , wherein:
 the A1 domain has an amino acid sequence at least 99% identical to SEQ ID NO: 22; 
 the A2 domain has an amino acid sequence at least 99% identical to SEQ ID NO: 23; 
 the ap domain has an amino acid sequence at least 99% identical to SEQ ID NO: 25; 
 the A3 domain has an amino acid sequence at least 99% identical to SEQ ID NO: 26; 
 the C1 domain has an amino acid sequence at least 99% identical to SEQ ID NO: 27; and 
 the C2 domain has an amino acid sequence at least 99% identical to SEQ ID NO: 28. 
 
     
     
       3. The protein of  claim 1  wherein the A2 domain comprises a glutamate or a valine residue at FVIII position 453, wherein the FVIII position is according to a FVIII protein sequence set forth as SEQ ID NO: 1. 
     
     
       4. The protein of  claim 1 , wherein the linker comprises an amino acid sequence between two and fifty amino acids in length. 
     
     
       5. The protein of  claim 1  comprising the amino acid sequence set forth as SEQ ID NO: 40 without the signal peptide of residues 1-19. 
     
     
       6. A nucleic acid encoding the protein of  claim 1 . 
     
     
       7. A pharmaceutical composition comprising the protein of  claim 1 . 
     
     
       8. A method of inducing blood clotting comprising administering an effective amount of a pharmaceutical composition of  claim 7  to a subject in need thereof. 
     
     
       9. The protein of  claim 3 , comprising the valine residue at FVIII position 453. 
     
     
       10. The protein of  claim 3 , comprising the glutamate residue at FVIII position 453. 
     
     
       11. The protein of  claim 1 , wherein the linker comprises an amino acid sequence set forth as SFSQNPPVLKRHQR (residues 761-774 of SEQ ID NO: 40). 
     
     
       12. The protein of  claim 2 , wherein the linker comprises an amino acid sequence set forth as SFSQNPPVLKRHQR (residues 761-774 of SEQ ID NO: 40). 
     
     
       13. The protein of  claim 1 , comprising an amino acid sequence at least 96% identical to SEQ ID NO: 40 without the signal peptide of residues 1-19. 
     
     
       14. The protein of  claim 13 , comprising an amino acid sequence at least 99% identical to SEQ ID NO: 40 without the signal peptide of residues 1-19. 
     
     
       15. The protein of  claim 14 , comprising the amino acid sequence set forth as SEQ ID NO: 40 without the signal peptide of residues 1-19 and further comprising a glutamate residue at FVIII position 453, wherein the FVIII position is according to the FVIII protein sequence set forth as SEQ ID NO: 1. 
     
     
       16. The protein of  claim 1 , wherein
 the A1 domain has the amino acid sequence set forth as SEQ ID NO: 22; 
 the A2 domain has the amino acid sequence set forth as SEQ ID NO: 23, further comprising a glutamate residue at FVIII position 453, wherein the FVIII position is according to a FVIII protein sequence set forth as SEQ ID NO: 1; 
 the ap domain has the amino acid sequence set forth as SEQ ID NO: 25; 
 the A3 domain has the amino acid sequence set forth as SEQ ID NO: 26; 
 the C1 domain has the amino acid sequence set forth as SEQ ID NO: 27; and 
 the C2 domain has the amino acid sequence set forth as SEQ ID NO: 28. 
 
     
     
       17. The protein of  claim 16 , wherein the linker comprises the amino acid sequence set forth as SFSQNPPVLKRHQR (residues 761-774 of SEQ ID NO: 40). 
     
     
       18. The protein of  claim 4 , wherein the linker comprises a PACE/furin processing sequence set forth as RHQR (SEQ ID NO: 245).